Project Summary Various G-protein-coupled receptors elicit distinct functional responses within a cell, even though they use the common diffusible second messenger cAMP. For instance, while stimulation of either ?-adrenergic receptors or E-type prostaglandin receptors leads to cAMP production, only ?-adrenergic receptors regulate cardiac myocyte contractility. The ability of a cell to distinguish between cAMP produced in the same cell can only be explained if engagement of different receptors generates distinct receptor-specific pools of cAMP. However, the underlying mechanisms responsible for creating compartmentalized cAMP are not completely understood. Compartmentalized cAMP signaling regulates cardiac contractility and thus is essential for normal functioning of the heart. Consistent with this, dysregulation of cAMP compartmentalization has been linked to several cardiovascular diseases, including cardiac arrhythmias, hypertrophy, and heart failure. Most previous studies have focused on activities of phosphodiesterases, the enzymes that breakdown cAMP, to explain cAMP compartmentation. However, several mathematical studies have predicted that PDE activity alone is not sufficient. These studies have suggested that the mobility of cAMP must be slower than free diffusion to prevent cAMP from reaching non-specific target proteins. We have recently demonstrated that the intracellular mobility of cAMP is markedly hampered by buffering mediated by mitochondria-associated protein kinase A. Now, a new computational study has predicted that, in addition to slow diffusion of cAMP, anatomically restricted spaces within a cell are key to hindering cAMP movement. In cardiac myocytes, mitochondria occupy 30% of the cell volume and are associated with constrained spaces through interactions with the sarcoplasmic reticulum and cytoskeletal proteins. The overall aim of this proposal is to explore the concept that the tight spaces associated with mitochondria regulate cAMP compartmentation. The tethering of mitochondria to the sarcoplasmic reticulum by the proteins, mitofusin-2 (MFN2), glucose-regulated protein 75 (GRP75), and phosphofurin acidic cluster sorting protein 2 (PACS2), creates tight spaces between these organelles. In the FIRST AIM of this study, we will test the hypothesis that the anatomically restricted spaces between mitochondria and the sarcoplasmic reticulum hinder cAMP movement and contribute to cAMP compartmentation. In cardiac myocytes, mitochondrial arrangement is regulated by microtubules and muscle LIM protein (MLP). Disruption of microtubules or MLP causes disorganization of mitochondria and alters mitochondrial morphology, thereby changing the cytosolic spaces associated with mitochondria. Thus, in the SECOND AIM, we hypothesize that cAMP compartmentation is hampered following mitochondrial derangement in microtubule-disrupted cells. To test these hypotheses, we adopt multipronged and complementary approaches to study cAMP compartmentation. Using a variety of advanced techniques, we will measure cAMP mobility, changes in cAMP levels within specific intracellular locations, changes in Ca2+ channel currents and intracellular Ca2+ transients, and test changes in functional responses, such as cell shortening, following stimulation of individual receptors. The goal of this proposal is to elucidate the fundamental mechanisms responsible for facilitating cAMP compartmentation. We believe that this approach may ultimately lead to the development of potential therapeutic strategies to overcome the burden of cardiac diseases.